A spraying method, a dropping method and the like are generally known as methods for preparing a sample to be analyzed for MALDI-MS by applying a matrix substance onto a substance to be analyzed so as to form a matrix film. Organic substances, which absorb the used laser beam with a certain wavelength range well, are used as the matrix substance, and examples include 1,4-bisbenzene, 1,8,9-trihydroxy anthracene, 2,4,6-trihydroxy acetophenone, 2,5-dihydroxy benzoic acid, 2-(4-hydroxy phenyl azo) benzoic acid, 2-amino benzoic acid, 3-amino pyrazine-2-carboxylic acid, 3-hydroxy picolinic acid, 4-hydroxy-3-methoxy cinnamic acid, trans-indoleacrylic acid, 2,6-dihydroxy acetophenone, 5-methoxy salicylic acid, 5-chloro salicylic acid, 9-anthracene carboxylic acid, indole acetic acid, trans-3-dimethoxy-hydroxy cinnamic acid, α-cyano-4-hydroxycinnamic acid, 1,4-diphenyl butadiene, 3,4-dihydroxy cinnamic acid and 9-amino acridine.
Different matrix substances from among these are used as a matrix substance appropriate for analysis depending on the substance to be measured or the properties and the ion polarities of the substance to be detected.
In recent years, a mass imaging method according to which the state where living body molecules and metabolite are localized on a piece of tissue can be two-dimensionally visualized using MALDI-MS has been attracting attention.
In the mass imaging method, the amount of the matrix applied to the substance to be measured (film thickness) greatly affects the results of mass imaging using MALDI. Therefore, it is important to form a matrix film having high reproducibility and uniformity, and a film formation technology for this has been demanded.
Application in accordance with a spraying method has been commonly used so far for the formation of a matrix film. In general, manual operation using a hand spray by a skilled worker is necessary in the spraying method, and furthermore, it is necessary to prepare a matrix solution to be applied. In addition, the state of crystals in the applied matrix, the amount of application and the reproducibility are greatly affected by the degree of skill of the worker and the environment within the room, and therefore, it is necessary to prepare a matrix solution for satisfying meticulous conditions by using many samples (including spare samples) every time a sample is applied, even for the same sample. Though there are automated spray devices, they are currently inferior to the skilled workers in terms of the quality of spraying.
In recent years, a vacuum vapor deposition method according to which the formation of a film of microscopic crystals is possible has been attracting attention as a new method for forming a matrix film (see Patent Document 1). The matrix films made in accordance with the vacuum vapor deposition method can be prevented from being affected by the inconsistency in the degree of skill of the workers or the environment within the room. However, most matrix substances are heated in a powdered state so as to be deposited from a vapor resulting from sublimation. It is difficult to secure sufficient reproducibility with the deposition from a vapor resulting from sublimation due to a change in the vapor pressure caused by the inconsistency in the heat conductance of the vapor source (a boat for heating or the like) or the difference in the purity even when the temperature for heating the vapor source is precisely controlled. Therefore, reproducibility is secured by monitoring during vacuum vapor deposition the amount of application for the matrix film (film thickness) that adheres the substance to be analyzed.
A method for indirectly finding the thickness of a film that has adhered to the substrate on which a film is to be formed from the results of measurement of the thickness of the film that has adhered to an element for measuring the film thickness (substrate for measuring the film thickness or crystal oscillator) that is different from the sample substrate has been known, whereby in order to monitor the thickness of the film that has adhered to the sample substrate that is an object of which film is to be formed during vapor deposition, this element to be measured for measuring the film thickness is attached in a location away from the sample substrate within the vacuum chamber (in a location that does not hinder the film formation on the sample substrate). Concretely speaking, a film thickness measuring method for measuring the film thickness from the change in the transmittance (amount of light that has transmitted) when a substrate for measuring a film thickness is used as an element to be measured, and this substrate is irradiated with light from the outside of the vacuum container (see Patent Document 2), and a method for measuring a film thickness from the change in the frequency due to the adhesion of a film to a crystal oscillator (see Patent Document 3) are known as examples.
Though there are some cases where measurement of the thickness of a thin film using the effects of interference is possible, such a measurement cannot be used when the formed film absorbs light for measurement, and the measurement using the effects of interference is difficult unless the film thickness is in an appropriate range.
Here, in order to confirm the film thickness after completion of vapor deposition, a contact step gauge can be used.